Human immunodeficiency virus type-1 (HIV-1) RNA transcripts are used to translate viral proteins, and are also selectively incorporated into nascen virions as the genetic material. HIV-1 is capable of packaging a single copy of dimeric, full-length genomic RNA (gRNA), ensuring correct propagation of its genetic information. Despite extensive study, the mechanism governing the selective incorporation of gRNA by HIV-1 remains poorly understood. The viral Gag protein is responsible for packaging gRNA via interactions with its 5 untranslated region (5UTR), specifically the Psi packaging signal. The 5UTR is known to adopt multiple conformations and dimerize, and this has been proposed to affect gRNA packaging. Gag has also been shown to adopt multiple conformations, fluctuating between bent and straight conformers depending on the presence of phospholipids and/or nucleic acids. The current model explaining genome packaging is that the nucleocapsid (NC) domain of Gag recognizes gRNA and targets it to the plasma membrane (PM) via the Gag matrix (MA) domain, nucleating further Gag oligomerization and leading to virus particle formation. A few Gag molecules likely interact with gRNA in an NC-only conformation to allow MA to bind the PM. However, a clear understanding of Gag conformation and specific gRNA interactions that lead to RNA packaging is lacking. I hypothesize that Psi-containing, dimeric gRNAs promote Gag interaction in an assembly-competent mode, leading to their preferential incorporation into virions. The following experiments will elucidate the RNA sequence and conformations that can alter Gag's binding mode and conformation, in order to establish how Gag interacts with these RNAs in a manner that leads to their selective encapsidation. This proposal seeks to answer the following outstanding questions: 1) What gRNA sequence and conformation are required for Gag interactions in the specific binding mode? 2) How does gRNA sequence and conformation affect the Gag conformational equilibrium? 3) Do these same gRNA sequences and conformations lead to selective gRNA packaging during infection? Elucidation of the molecular mechanism employed by Gag to successfully target and package its gRNA will facilitate future efforts at targeting this interaction with novel antiretroviral therapies.